CZ286766B6 - Attenuated virus strain, process of its preparation, vaccine, process of its preparation, cellular clone and use of the attenuated strain - Google Patents

Abstract

In the present invention there is disclosed an attenuated virus strain causing pig disease known as pig reproduction and respiration syndrome (PRRS) that completely corresponds to a deposit with number I-1642 in CNCM Institut Pasteur deposited on November 23, 1995. In the present invention there is also indicated a cellular clone C8 CNCMI-1643 in which the PRRS virus is capable of replication up to high titer 10e5 to 10e7 TCIDi50/ml. In the present invention there are further claimed a process for preparing such virus, a vaccine containing thereof and its preparation process as well as the use of the attenuated virus strain for preparing the vaccine.

Description

Attenuated virus strain, method of its preparation, vaccine, method of its preparation, cell clone and use of attenuated virus strain

Technical field

The present invention relates to a novel attenuated strain of a virus causing pig disease known as porcine reproductive and respiratory syndrome (PRRS). The attenuation and replication procedures of a virulent strain using a new monkey kidney cell clone allow the preparation of vaccines and diagnostic kits that allow early diagnosis of PRRS and effective preventive treatment of the disease.

BACKGROUND OF THE INVENTION

In 1987, pig disease was first detected in North America, which at that time was defined as 'Major Swine Disease' or MSD, and later known as 'Pig Infertility and Respiratory Disorder Syndrome', or SIRS. A very similar syndrome was first detected in Central Europe in 1990 and later spread to other European countries, including Spain. Initially, the disease was named in Europe as "Epidemic Abortion and Pig Respiratory Syndrome" or PEARS and finally as "Porcine Reproductive and Respiratory Syndrome" or PRRS. This name has been accepted worldwide for this disease.

It is already known that the etiological agent of PRRS is a small encapsulated RNA virus, first isolated in the Netherlands and named as Lelystad virus. It has been suggested that the virus belongs to the Arteviridae group. This virus has been described in PCT patent application WO-92/21357 and European patent application EP-B-0587780 (Stichting Centraal Diegeneeskundig Instituut), the latter being derived from the first. For the purpose of this application, an isolate of the above virus was deposited with the Institut Pasteur of Paris, number 1-1102.

The North American virus type was isolated almost simultaneously with the European virus type as described in PCT patent application WO 93/03760 (Collins et al.) And in European patent application EP-A0529584 (Boehringer Ing.). For the purposes of this application, the isolate of the above virus was deposited in American Type Culture Collection (ATCC), No. VR-2332.

The European and North American virus types are clearly different, not only with regard to serological reactivity, but also with regard to the degree of homology of the nucleotide sequence of the significant RNA fragments. On the first two pages of European patent application EP-A-0676467 (Akzo) there is a detailed description of these differences, with extensive references to the literature. In the aforementioned patent application, it is considered that the European type and the American type of viruses clearly diverged a long time ago. As a result, it can be expected that an eventually effective vaccine against one of these types will be little or no effective at all against the other type.

Various strains were isolated from both European and American virus types. Each strain has its own specific characteristics, and several strains have been the subject of a patent application. For example, PCT patent application WO-93/07898 (Akzo) describes a European strain, and a vaccine derived therefrom, deposited at CNCM (Institut Paster), No. 1-1140. PCT patent application WO93 / 14196 and European patent application EP-A-541418 (Rhone-Merieux), both derived from the application of the same priority, describe a new strain isolated in France, deposited by CNCM (Institut Pasteur), No. 1-1153. European patent application EP-A-0595436 (Solvay) describes a new strain of the American type, more viruent than previously described, and a vaccine thereof. This strain has been deposited with the ATCC, but the accession number is not detailed in this application. Finally, Spanish patent application ES-A-2074950 (Canamid Iberica) describes a so-called "Spanish

Strain ', which is different from other European and American tribes. This "Spanish strain" was deposited at the European Animal Cell Culture Collection (EACCC), No. V93070108.

In conclusion, it seems clear that the etiological agent of PRRS shows many varieties and to effectively combat this disease, vaccines of various types are needed depending on the strain of virus that infects pigs.

In swine, the disease is characterized by loss of appetite, anorexia, reproductive disorders (abortion, premature birth, birth of dead or weak piglets and fetal death, with or without mummification). Some infected pigs may die. Less common symptoms are transient wet discoloration of the ears, abdomen or vulva; according to this, the disease was first known as 'blue abortion' in the Netherlands and 'blue ears' in Great Britain. Dyspnoea or tremor may be observed in newborn piglets, whereas posterior paresis and ataxia are more common in older piglets. At the peak of the outbreak, mortality during the first days is limited, but may reach as much as 80% in ten-day piglets. Infected feeding piglets drink less and exhibit more respiratory problems.

The incubation period of the disease is very variable, ranging from 5 to 37 days (I.B. Robertson Europ. Comm. Comm. Seminar on PRRS, 11: 4-5, Brussels, 1991). Sometimes the disease spreads very slowly, but if the farm is affected, the disease may persist for several months (B. Thacker, Int. Symp. On SIRS, St. Paul / Minnesota, 1992).

Anti-virus antibodies were detected by the immunoperoxidase technique (immunoperoxidase monolayer assay, herein IPMA) on day 6 post-infection, as described by Wensvoort, G., et al. (The Vet. Quart. 13: 121-130, 1991). Antibody titers can reach 1/20,000 five days later, and usually persist for more than 12 months. However, some pigs become seronegative 4-5 months later, as described by V. Ohlinger et al., Meredith, M. De. Pig Dis. Info. Center Cambridge, 12/1992. These authors were able to isolate virus after infection from various organs, with a virus titer reaching 10 ⁴ TCID ₅₀ (tissue culture infectious dose 50%) 6 days after infection in lung, serum, plasma and blood cell homogenates. This shows that the virus and antibodies can persist for several weeks, moreover, it is well known that animals that survive the outbreak of disease can be a source of infection for susceptible animals. Viremia can be detected from day 1 after infection and can last up to 56 days, although it is usually shorter.

In the hematogenous spread of the virus, the virus can reach the placenta of pregnant sows. It has been shown that the virus can cross the placenta and cause fetal death. Maximum fetal sensitivity occurs during the third third of gestation. In addition, the virus is capable of replicating in the fetus without causing death. However, the virus has never been isolated from mummified or autolysed fetuses.

In piglets, the onset of the disease occurs when the level of colostral maternal antibodies is reduced. In live-born piglets from sows infected in the last third of pregnancy, some cases of piglets with anti-virus antibodies were observed before colostrum secretion was observed. Usually, these animals show viremia at birth (C. Terpstra et al, Vet. Q. 13: 131 136, 1991).

Despite the destruction of a large number of macrophages, the immunosuppressor activity of PRRS-causing virus has not been clearly established. However, associated secondary infections are common and cause serious economic losses on pig farms.

Currently, the PRRS virus has been found in most countries with a significant pig population.

Currently, PRRS is one of the most important diseases affecting pigs due to secondary losses, both direct and indirect, caused by secondary agents after infection with PRRS virus.

-2GB 286766 B6

The use of inactivated vaccines reduced in porcine alvolar macrophage (PAM) cultures gives acceptable results at laboratory level, but efficacy in field conditions depends, in part, on environmental conditions and the breeding of vaccinated animals.

One of the problems that hinders the acquisition of immunological products against PRRS virus is the limited availability of stable substrates for virus replication.

So far, the PRRS virus could only be applied in porcine alveolar macrophage (PAM) cultures (Wensvoort G., et al., In The Vet. Quart. 13: 121-130, 1991). The need to use healthy pigs of a certain age to obtain these macrophages implied serious obstacles. In addition, susceptibility to viral infection was not guaranteed in the obtained PAM since cell substrates derived from different animals are already different. This identified a major obstacle to the production of antigen batches of constant and homogeneous quality and each batch had to be evaluated to determine its sensitivity.

For all these reasons, the reduced availability of a stable continuous cell host seriously hindered the study of strategies for obtaining mutants based on PRRS virus replication in cell substrates and the selection of attenuated varieties.

One of the main problems to be solved is the need to neutralize the virulent virus avoiding its replication in PAM, since the replication of the virus causes their destruction.

Accordingly, adaptation of the virus to a stable substrate derived from a transformed cell line will provide a suitable tool both for obtaining attenuated mutants and for preparing inactive vaccines, eliminating PAM dependence and variability.

PCT patent application WO-94/18311 (Miles) proposes the propagation of certain PRRS virus strains in a unique clone, designated as clone 9009B, derived from the African green monkey kidney cell line, designated MA-104 (M) by a patent application, derived from a commercial cell line. known as MA-104. In the patent application the accession number of said unique clone is not given and therefore the evaluation of the application and the reproduction are difficult.

SUMMARY OF THE INVENTION

The present invention is based on an attenuated strain of swine disease virus known as porcine reproductive and respiratory syndrome, PRRS, which fully corresponds to deposit number 1-1642 of the CNCM Institut Pasteur, deposited on November 23, 1995.

The present invention also provides a vaccine for the protection of pigs against a disease known as porcine reproductive and respiratory syndrome, PRRS, comprising a viral strain as described above and / or at least one viral antigen obtainable from said strain.

The present invention also provides a C8 cell clone derived from the MA-104 monkey kidney cell line, which has 10 ⁵ to 10 ⁷ TCID ₅ o / ml and deposited with the CNCM Institut Pasteur under deposit number 1-1643.

The invention also provides the use of the attenuated virus strain described above for the production of a vaccine against swine disease, known as PRRS.

The attenuated strain of PRRS virus object of the present invention was derived by attenuating a virulent strain isolated from infected pigs from a Spanish farm. In order to fulfill the conditions of the description, this attenuated strain was deposited in the Collection Nationale de

-3 CZ 286766 B6

Cultures de Microorganismes (CNCM) of the Institute Pasteur, with accession number 1-1642. Date of deposit is 23.11.1995.

Attenuation of the virulent strain and replication of the attenuated strain were performed by serial passage in a cell clone, which is also an object of the present invention, which was designated by the authors as Clone-8. This clone is derived from a commercial monkey kidney cell line known as MA-104. Also to meet the conditions of the description, clone-8 was deposited, on the same day as above, in the CNCM of the Pasteur Institute, with accession number 1-1643.

First, the commercial cell line MA-104 was cloned for selection and isolation of clone 8. This cloning was performed by suspending cells in a suitable culture medium (e.g., Earl's Minimum Essential Medium (Earle's MEM) with Fetal Bovine Serum (FBS)), placing the suspension at various concentrations, selection and trypsinization of the clones and their expansion in culture vessels. Clones obtained in this way were sequentially cloned using the techniques described until well differentiated clones were obtained.

After removal of clones that showed irregular behavior or which were difficult to amplify, the selected clones were tested for susceptibility to infection to the initial virulent PRRS virus strain. Clone 8 was selected because of its high sensitivity to viral strain replication (high TCID ₅₀ ) and the reproducibility and consistency of the virus yield obtained.

The virulent strain was attenuated by sequential replication in clone-8 cultures, preferably at 34 ° C. The content of infectious virus particles was determined to assess relicative viability and the cytopathic effect (CPE) was examined to determine the degree of adaptation. According to the results obtained, the virus is capable of replicating in clone-8 without loss of viability for at least 20 passages, and attenuation produces a virtually harmless virus that retains its antigenic activity.

As a result, the attenuated PRRS virus strain of the present invention is obtained in a stable and industrially reproducible manner, which facilitates its use for obtaining PRS vaccines and PRRS diagnostic kits.

Comparative tests between groups of pigs infected with either a virulent strain or an attenuated strain have clearly shown that the attenuated strain is harmless to the animals infected with it. On the other hand, the attenuated strain object of the present invention exhibits high replication efficacy in seronegative pigs and is able to induce seroconversion in animals inoculated with a dose of 200 TCID 50 by intramuscular. Antibodies elicited in this manner persist for at least 80 days.

Furthermore, the attenuated strain object of the present invention is an excellent basis for the preparation of vaccines for preventive treatment of pigs against PRRS. Such vaccines may be prepared using well known methods by those skilled in the art and in various conventional forms such as aqueous dispersions, oil emulsions, liposome compositions, lyophilized forms, etc. The vaccine compositions may be supplemented with various adjuvants such as immunostimulants, emulsifiers, stabilizers, etc. The vaccines may be administered intramuscularly or subcutaneously, intranasally, intratracheally, cutaneously, percutaneously or intecutally.

Effective doses of the vaccine will vary widely, in a preferred form ranging from 10 ² to 10 ⁶ TCID 50 of the attenuated strain object of the present invention.

The obtained vaccines, also an object of the present invention, can also be formulated as polyvalent vaccines, together with another live or inactivated swine virus, or together with a live or inactivated bacterium.

-4GB 286766 B6

As will be appreciated by those skilled in the art, vaccines comprising the viral antigens derived from the viral strain of the present invention, for example, vaccines comprising said strain in completely inactivated form, can also be prepared using any conventional method, e.g. or RNA fragments of said strain and the like.

The attenuated strain object of the present invention can also be used to prepare suitable diagnostic kits - using conventional techniques - containing antigenic elements capable of detecting antibodies in seropositive animals. For example, IPMA procedures for detecting PRRS antibodies may include the following steps:

a) Adaptation of the attenuated strain of the present invention to a stable cell culture, preferably clone-8, on a culture microplate such that each well is infected with about 20-40 infectious particles.

b) Fixing the infected cells to the solid phase using known fixants.

c) Detecting porcine serum antibodies by incubating them in the microplate and then staining said antibodies on the microplate with IMPA techniques.

Overview of the drawings

Two pages with four figures are attached to and included in the present description. The figures are illustrative and do not limit the present description.

Figure 1 shows a two-dimensional figure representing the development of rectal temperature in pregnant sows inoculated in an intranasal manner with an attenuated strain object of the present invention.

Figure 2 is a two-dimensional figure representing the development in weight of piglets born to a sow inoculated with an attenuated strain object of the present invention.

Figure 3 is a two-dimensional figure representing the kinetics of colostral antibodies as determined by IPMA, in the offspring of sows vaccinated with the attenuated strain object of the present invention. Low titers in the offspring of sows 1 and 10 at birth are due to the fact that some piglets have not yet sowed colostrum at the time of blood collection.

Figure 4 shows a three-dimensional image representing the development of antibody response in piglets inoculated by intramuscular route 200, 2000, and 20,000 TCID _{50 of the} attenuated strain object of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Several examples of procedures are shown to illustrate the present invention in more detail. Such examples do not limit the scope of the invention.

Example 1: Obtaining cell clones from a stable monkey kidney cell line MA-104.

Six passages of the selected PRRS virulent strain were performed using the MA-104 monkey kidney cell line provided by the European Animal Cell Culture Collection (EACCC) accession number 85102918, in a static cell culture monolayer cultured in plastic.

Culture vessels in Earle's MEM medium supplemented with 10% fetal bovine serum (FBS), at 37 ° C and without CO ₂ replenishment. Virus harvest was performed 6-7 days after inoculation at each passage.

To study viral harvest gains, the cell line was inoculated with a virulent strain at various strengths of infection (here MOI). Virus harvest yields were low (between 10 ³ and 10 ⁴ TCID ₅₀ / ml) and insufficient for use in vaccine production, even after four adaptation passages.

In these experiments, it was observed that only a portion of the infected cells were susceptible to the virulent virus, while the remainder of the cells remained refractory to the infection. For this reason, the cell line was cloned in order to select that cell population that is completely sensitive to the virulent strain.

The cloning of the selection was performed as follows:

The cell line suspension was diluted in Earle MEM with 20% FBS and various dilutions were placed in a 96 well microplate (NUNC ^R ). The plates were incubated for 8 days at 37 ° C in 5% CO ₂ . On day 3, wells containing only one cell were identified microscopically and were trypsinized on day 8. In this way, 44 clones were obtained. Then, the obtained clones were expanded in culture flasks until a suspension of 5 x 10 ⁷ cells / ml was obtained. These cells were then cloned a second time and needed using the same procedure.

After the third cloning, 44 obtained clones were expanded until 25 ml of cell suspension of each clone containing 6 x 10 ⁶ cells / ml was obtained. The medium used in this process contained 20% FBS.

These clones were further selected according to their growth characteristics and their viability.

Example 2: Selection of a clone-8 cell clone

The clones obtained in the previous example were evaluated for their growth efficiency, and those that showed amplification problems, irregular morphology or were difficult to maintain at 37 ° C were removed. 35 clones were removed after this previous selection and the remaining 9 clones were selected.

The selected cell clones were infected with the virulent strain PRRS virus in eight virus passages (P-8) and PAM cultures (as described by Bloemberg, M. et al., Vet Microb. 42: 361-371, 1994). The adaptation process for cell clones was performed before: the virus was replicated at 37 ° C for 3 passages by keeping an 80-90% confluent monolayer in contact with the virus suspension for 6 days and then frozen at -80 ° C and thawed 24 hours later.

Earle MEM supplemented with 10% FBA and gentamycin (0.4 mg / ml) was used as an infectious medium. Neither fungal compounds nor yeast compounds were used. Each virus harvest obtained in this way was titrated in the corresponding cell clone. After analysis of the results, it was concluded that 9 clones were susceptible to viral infection, with a titer equal to or greater than 10 ^{4 2} TCID ₅ o / ml. The results are shown in Table 1.

-6GB 286766 B6

Table 1: Sensitivity of the obtained cell clones to the selected virulent strain

Clone or cell line Titer as TCID ₅ o / ml uncloned cell line 10 ³ clone 1 10 ⁵⁷⁵ clone 2 10 ⁴⁵ clone 3 10 ⁵² clone 4 10 ⁵³ clone 8 10 ⁶ clone 29 10 ⁵⁷⁵ clone 30 10 ⁵³⁸ clone 41 10 ⁵³⁸ clone 44 10 ⁴²

As can be seen in Table 1, an uncloned cell line exhibits a very low sensitivity to the virus and therefore its use as an antigen to obtain an effective vaccine does not seem possible. Table 1 also shows that some clones, especially clone 8, are more sensitive to the virus than an uncloned cell line. Clone-8 is particularly worthy of recording. Titers greater than 10 ⁶ TCID ₅ o / ml can be obtained when harvesting the virus from clone-8.

In accordance with the results described above, clone-8 was selected. Several tests were performed for the reliability stage of this clone. In various tests, viral harvest titers were highly reproducible, with values between 10 ⁵ and 10 ⁷ TCID ₅ o / ml.

Example 3: Obtaining an attenuated virus strain

The attenuated virus strain of the present invention was obtained by replicating the selected virulent strain at 34 ° C in clone-8 cell cultures.

Virus-infected monolayers of clone 8 cells were maintained at 34 ° C until CPE was developed. CPE was detected 24 and 48 hours later than in cultures maintained at 37 ° C. However, no significant differences were found in the CPE properties developed at both temperatures.

The resulting viral harvests were titrated using clone-8 cell mono layers. Furthermore, the identity of the virus was verified by IMPA.

The virus was inoculated into a 75 cm ² clone-8 cell monolayer around confluence and allowed to adsorb at 34 ° C for 2 hours. Then, the infectious medium was added to the monolayer. The infectious medium was Earle modified minimal essential medium supplemented with 10% FBS and pre-heated to 34 ° C.

cm ² flasks were placed at 34 ° C in an incubator and checked daily until CPE was observed. When 80-95% of the cell monolayer showed CPE, usually between 5 and 7 days after infection, viral harvesting was performed. The viral harvest was then centrifuged at 2000 rpm and the supernatant was titrated to determine the virus titer (TCID ₅₀ o / ml).

Using the method described above, 20 passages were performed. Virus content was evaluated at passages P.1, P.5, P.10 and P.20. The results show that the virus can be replicated in the cell monolayer of clone-8 at 34 ° C, without any loss of viability, at least until passage P.20 (Table 2).

-7EN 286766 B6

Table 2: Development of virus propagated in clone-8 at 34 ° C from passage P.l to passage P.20.

Arcade in clone-8 Virus content in TCID ₅ o / ml P.l 10 ⁵⁵ P.5 10 ⁵⁷ P.10 10 ⁵⁵ P.15 10 ⁵⁶ P.20 10 ⁶²

Further results confirmed that clone-8 cell monolayer can be infected at an MOI of 0.001.

Experiments carried out according to the following example showed that the P.20-derived virus strain was harmless to pigs.

Example 4: Biological characterization of the attenuated viral strain and the effects of vaccination with this strain

The initial viral strain used to obtain the attenuated virus strain of the present invention is a virulent strain. The main effects on pregnant sows are premature births and weak, dead and / or mummified newborn piglets. Infected sows also show depression and a 3-5 day period of mild anorexia on day 4-5 after infection.

Four pregnant sows (designated 01, 02, 03 and 06) were infected intranasally with 10 ⁶⁶ TCID _{50 of the} initial virulent strain. Two uninfected swine (labeled 73 and 74) were used as controls. The results are shown in Table 3.

Table 3: Effects on offspring of pregnant sows inoculated with virulent virus

Svině č. Inoculation with virulent virus Number of piglets born Number of dead piglets until weaning live dead mummified 01 + 10 2 0 2 02 / + 15 Dec 0 0 4 03 / + 6 5 0 2 06 / + 6 6 0 1 73 - 13 0 0 0 74 - 10 0 0 0

Only infected swine gave birth at the expected date. The infected sows had 2, 0 5 and 6 stillborn piglets and 2, 4, 2 and 1 weak piglets, respectively. Weak piglets died a few days after birth. At weaning, an average of 11 piglets from each control were alive. Only an average of 6.5 piglets from each infected swine were alive at weaning. At weaning, the average weight of piglets was 4.621 g (piglets from infected sows) and 5.365 g (piglets from control sows). Virulent PRRS virus was isolated from homogenized lungs of weak piglets. After challenge, both the infected swine and their offspring seroconverted.

Biological characterization of the attenuated virus strain of the present invention, according to specific tests, was performed using pigs of both sexes. A harmless test was performed on 3 PRRS seronegative pigs from a small farm where PRRS infection was never detected. The sows were inoculated in the last third of pregnancy when the virus sensitivity was maximal. A dose of 10 ⁶ TCED ₅ o / swine of the attenuated virus was administered intranasally

-8GB 286766 B6 in a manner between day 78 and 93 of pregnancy. After virus inoculation, the physiological constants of the three sows remained unchanged and the rectal temperature was within normal (see Figure 1). The three pigs also showed the expected data. The results obtained are summarized in Table 4.

Table 4: Effects on offspring of pregnant sows inoculated with attenuated virus

Swine No. Atte inoculation - Number of piglets born Number of deaths due to nuanced virus live dead mummified PRRS until weaning

1 + 13 0 3 0 2 + 4 1 0 0 10 + 16 0 1 0

The three sows gave birth to 13, 4 and 1 piglets, respectively. Vitality of newborn piglets was considered normal. However, several mummified piglets were found in the offspring of two sows. This can be considered normal due to the large litters of these pigs. Piglet weight development was in all cases considered normal (see Fig. 2) and was within normal limits (45 days) until the end of observation. Symptoms of weakness and disease that may be related to PRRS virus infection were not observed in any piglet. Furthermore, PRRS virus was not detected in blood or serum samples from newborn piglets.

Similarly, PRRS virus was not detected in blood and serum samples from pregnant sows 21-36 days after virus inoculation. All these facts confirm the harmlessness of the attenuated PRRS strain in pregnant sows, which are the most sensitive category for infection with wild-type virus. All serum samples from newborn piglets from inoculated pregnant sows were negative when examined for PRRS virus and PAM cultures using conventional techniques. The three pigs inoculated with the attenuated virus had humoral antibodies of IPMA 1/480 titers at day 45 postpartum, with stable stability with slight deviations from that day. The sows were seropositive as early as 21 days after inoculation. As seen in Figure 3, piglets from inoculated sows were seropositive after colostrum suction and remained seropositive for at least 75 days of age.

Another harmless test was performed in a group of 12 pregnant sows in the last third of pregnancy. Eight sows were vaccinated intramuscularly with 39 doses of vaccine (10 ⁵ TCID ₅₀ ) and the remaining four sows were used as controls to assess the effects of the attenuated strain on reproduction parameters. No changes were observed in the physiological parameters of the vaccinated sows showing the expected data. The results are shown in Table 5. As can be seen from this table, both the eight inoculated sows and the four control sows showed a normal number of piglets. The piglets also had normal viability.

-9EN 286766 B6

Table 5: Effect of attenuated virus inoculation and reproduction parameters in pregnant sows in the last third of pregnancy

No swine vaccinated Deviation from expected delivery date (in days) Number of live piglets (A) Number of piglets weighing less than average weight group (B) Number of piglets at birth of the weak (C) Number of stillborn piglets (D) Number of mummified piglets (E) Total number of piglets (F) 86 Yes K +) 11 1 0 1 0 12 94 Yes 0 9 0 0 0 1 10 96 Yes K +) 13 2 1 0 0 13 102 Yes 2 (+) 13 1 0 1 2 16 116 Yes 0 10 0 0 1 0 11 121 Yes K +) 11 0 0 1 0 12 143 Yes i (-) 14 2 1 2 1 17 151 Yes 2 (+) 8 0 0 0 0 8 76 No 2 (+) 7 0 0 0 2 9 79 No 0 13 2 1 1 0 14 81 No I (-) 9 0 1 0 0 9 83 No LA) 10 1 0 1 1 12

A includes BaC: F = A + D + E

The attenuated virus object of the present invention replicates efficiently in PRRS seronegative piglets. This is demonstrated by the fact that a small dose such as 200 TCID ₅₀ administered intramuscularly can replicate and induce seroconversion in pigs. As seen in Figure 4, induced antibodies persist for at least 52 days in inoculated animals.

The attenuated virus of the present invention did not spread to four uninoculated sentinel piglets placed together with a group of eight intramuscularly inoculated piglets. The fact that the virus is not transmitted to unvaccinated animals illustrates the suitability of the attenuated strain for the vaccine. In addition, no leukopenia or other clinical symptoms were observed in vaccinated pigs to indicate that the attenuated virus is also harmless when administered intramuscularly. On average 86% of the inoculated piglets became seropositive as early as 11 days after inoculation.

The attenuated virus of the present invention induces a protective immune response in vaccinated pigs which prevents the clinical effects of infection with a virulent PRRS strain. Thus, no clinical signs were observed in 75% of four week old vaccinated piglets when infected with a virulent virus. On the other hand, as shown in Table 6, 80% of unvaccinated control piglets had a significant increase in rectal temperature after challenge. Furthermore, at necropsy performed 19 days after challenge, the vaccinated piglets showed significantly less lung lesion than the control unvaccinated piglets. Similarly, after challenge, virulent virus was found in only 25% of vaccinated animals, whereas in unvaccinated control animals, virulent virus could be found in 80% at least 12 days after infection.

Table 6: Hyperthermia in vaccinated and control piglets after infection

Number of piglets with hyperthermia / total number of piglets Number of days with hyperthermia Vaccinated piglets 1/4 1 (intramuscular) Non-vaccinated piglets 4/5 8

-10GB 286766 B6

Information on depositing microorganisms

Under the Budapest Treaty, both the viral strain and the clone 8 of the present invention were deposited with the Intemational Authority of the Collection of National Cultures of Microoganismes (CNCM) Institut Pasteur, Paris, (France)

Identifier of the reporter

VP-046-BIS

CNCM no.

Date saved

1-1642

23.11.1995

Aperture-8

1-1643

23.11.1995

These deposits are available to the public under the conditions specified in the Budapest Treaty. This cannot be interpreted as a license for carrying out the subject matter of the present invention, which would be a violation of the rights of the applicant of the present invention.

Claims (15)

PATENT CLAIMS An attenuated strain of swine disease virus known as porcine reproductive and respiratory syndrome (PRRS) CNCM 1-1642. A vaccine for the protection of pigs against a disease known as porcine reproductive and respiratory syndrome (PRRS), which comprises the viral strain claimed in claim 1 and / or at least one viral antigen obtainable from said strain. The vaccine of claim 2, wherein the viral antigen is an inactivated virus. The vaccine of claim 2, comprising between 10 ² and 10 ⁶ TCID 50 of the attenuated virus strain. The vaccine of claim 2 or 3, wherein the vaccine dose comprises an amount of viral antigen that is equivalent to the amount produced between 10 ² and 10 ⁶ TCID _{50 of the} attenuated virus strain. A vaccine according to any one of claims 2 to 5, which comprises further immunostimulatory adjuvants and / or emulsifiers and / or stabilizers. A C8 CNCM 1-1643 cell clone derived from the MA104 monkey kidney cell line, wherein the virus of claim 1 is capable of replicating up to a high titer of 10 ⁵ to 10 ⁷ TCID 50 / ml. 8. A method of preparing an attenuated PRRS virus strain according to claim 1, wherein the virulent strain is modified by sequential passage in a cell clone according to claim 7. The method according to claim 8, characterized in that a temperature of 34 ° C is maintained during successive passages. The method of preparing a PRRS vaccine according to claim 2, wherein the viral strain of claim 1 propagates in the cell clone of claim 7. -11EN 286766 B6 Method according to claim 10, characterized in that the virus strain propagates at a temperature of 34 ° C. Method according to either of claims 10 or 11, characterized in that the obtained attenuated strain is processed into an aqueous dispersion, an oil emulsion, a liposome-type composition or a lyophilized composition, optionally together with an auxiliary agent and / or emulsifier and / or stabilizer. Process according to one of Claims 10 to 12, characterized in that the strain obtained is treated by heat treatment or by a chemical process until complete inactivation. Use of the attenuated viral strain according to claim 1, for the manufacture of a vaccine against a disease of pigs known as PRRS. Use of the attenuated viral strain according to claim 1 for the manufacture of kits for detecting PRRS disease in pigs.